Metabolism of estradiol in liver cell culture Differential responses of C 2 and C 16 oxidations to drugs and other chemicals that induce selective species of cytochrome P 450

Metabolism of estradiol in liver cell culture.

Differential responses of C-2 and C-16

oxidations to drugs and other chemicals that

induce selective species of cytochrome P-450.

J Schneider, … , S Sassa, A Kappas

J Clin Invest.

1983;

72(4)

:1420-1426.

https://doi.org/10.1172/JCI111098

.

The oxidative metabolism of estradiol (the natural estrogen 2,3,5(10)-estratriene-3,17

beta-diol) at positions C-2 and C-16 was examined in primary cultures of chick embryo liver cells

using estradiol which was labeled with 3H specifically at either the C-2 or C-16 position as

the substrate. Oxidation of estradiol by the cultured liver cells was assessed by the release

of 3H which accumulated as 3H2O in the culture medium; both C-2 and C-16 oxidative

reactions were detectable in the liver cell cultures by this technique. When incubated with a

concentration of estradiol substrate close to the Michaelis constant (Km), approximately

45.8 pmol [2-3H]estradiol and 5.0 pmol [16-3H]estradiol/mg protein per minute underwent

oxidative metabolism in untreated cells. Total amounts of oxidized product formation after 2

h of incubation were 28 and 5 pmol/mg protein for C-2 and C-16 oxidation, respectively.

Treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide significantly

increased oxidation at C-16 (1.9-fold and 2.6-fold greater than control values, respectively),

whereas no significant change in C-16 oxidation was observed after treatment of the

cultures with 3-methylcholanthrene, benzo[a]pyrene, or benz[a]anthracene. The latter

chemicals, however, were found to increase the extent of oxidation at C-2 significantly (i.e.,

1.5-2.2-fold increases over control values). The increase in C-2 oxidation after treatment of

cultures with phenobarbital or 2-propyl-2-isopropylacetamide was significantly less than that

observed for oxidation at C-16. The […]

Research Article

Find the latest version:

Metabolism of Estradiol

in

Liver Cell

Culture

DIFFERENTIAL RESPONSES OF C-2 AND C-16 OXIDATIONS TO

DRUGS AND OTHER CHEMICALS THAT INDUCE SELECTIVE SPECIES OF CYTOCHROME P-450

JILL SCHNEIDER, SHIGERU SASSA, and ATTALLAH KAPPAS, The Rockefeller University Hospital, New York 10021

A

B

S T R A C T

The

oxidative metabolism of estradiol

(the

natural

estrogen

2,3,5(10)-estratriene-3,17,-diol)

at

positions C-2

and C-16 was examined

in

primary

cultures

of

chick

embryo

liver cells using estradiol

which was labeled with

3H specifically

at

either the

C-2 or C-16 position

as

the substrate. Oxidation of

estradiol by the cultured liver cells was assessed

by

the

release

of

3H

which

accumulated

as

3H20

in

the culture

medium; both

C-2 and C-16 oxidative

reactions were

detectable

in

the

liver

cell

cultures

by

this

technique.

When

incubated with a concentration of

estradiol

sub-strate

close

to

the Michaelis

constant

(K.),

-45.8

pmol

[2-3H]estradiol

and 5.0

pmol

[16-3H]estradiol/mg

protein per minute underwent oxidative metabolism

in

untreated cells.

Total amounts

of oxidized

product

formation after

2

h

of

incubation were 28 and 5

pmol/

mg protein

for

C-2

and C-16

oxidation,

respectively.

Treatment

of cultures

with

phenobarbital

or

2-propyl-2-isopropylacetamide

significantly

increased

oxidation

at

C-16

(1.9-fold and 2.6-fold

greater than control

val-ues,

respectively),

whereas no

significant change

in

C-16

oxidation

was

observed after

treatment

of the

cul-tures

with

3-methylcholanthrene,

benzo[a]pyrene,

or

benz[a]anthracene.

The

latter

chemicals, however,

were

found

to

increase the

extent of

oxidation at C-2

significantly

(i.e.,

1.5-2.2-fold

increases over

control

values). The

increase in C-2 oxidation after treatment

of cultures with phenobarbital or

2-propyl-2-isopro-pylacetamide

was

significantly

less than that observed

for oxidation at C-16. The apparent Km values for these

oxidations in control cultures

were 23.5

and

30.3

_;&M

Dr. Schneider wassupported by a Burroughs Wellcome

ScholarshipinClinicalSciences atThe Rockefeller

Univer-sity Hospital.

Receivedforpublication26July1982andinrevised form 21 June 1983.

for C-2 and C-16 oxidation, respectively;

correspond-ingmaximum

velocity

(V..ax)

values

were 119

and

11.7

pmol/mg

protein per minute,

respectively. These data

indicate that the

C-2

and C-16 oxidations of estradiol

take place

in

cultured

avian

hepatocytes and that the

extent

of metabolism

at

these

positions on

the

hormone

molecule

can

be altered by

chemicals, such

as

drugs

and polycyclic

aromatic

hydrocarbons, which induce

distinctive

species

of cytochrome P-450

in

the liver.

INTRODUCTION

Cytochrome

P-450

catalyzes the oxidative metabolism

of

a

wide variety of structurally diverse chemicals

in

the liver and other tissues (1). Multiple forms of this

microsomal

hemeprotein

exist

that

differ with

respect to

their substrate specificity and their degree of

in-ducibility and inhibition by various agents (2-4).

Ste-roid

hormones

represent a major

class

of endogenous

compounds metabolized

in

the

liver by cytochrome

P-450-dependent monooxygenases

in

mammals and other

species. For

example, androgens, such

as testosterone

and androstenedione, are biotransformed not only by

reductive metabolism at the C4-5 double bond,

but

also by hydroxylation reactions catalyzed by

cyto-chrome P-450 in rat liver (5, 6); the substrate

speci-ficity and the regulatory mechanisms of these

oxida-tions

have been extensively examined (7, 8).

Differ-ential inducibility of P-450-dependent steroid

oxidations has also been demonstrated in trout liver

microsomes

(9).

Wedescribe in this

paper the oxidative

metabolism

of estradiol (the natural estrogen

2,3,5(10)-estatriene-3,17#3-diol)

at

the C-2 and C-16 positions

in

primary

monolayer cultures of chick embryo liver cells

incu-bated

in a

chemically defined medium. Evidence

is

avian

embryo

liver are

cytochrome P-450-dependent

and

that theextentof the C-2and C-16 oxidationscan

be selectively altered by

drugs and other agents that

induce different

species of

cytochrome

P-450. These

findings

may have

potential

clinical

implications,

be-cause C-2 and C-16 oxidations of estradiol represent

the

primary metabolic

pathways

of estradiol in hu-mans and because their differential

inducibility by

drugs, environmental chemicals,

and nutritional fac-tors thus could alter the patterns of

endogenous

hor-mone biotransformation in man.

METHODS

Preparation of

[2-3H]estradiol

and[16-3H]estradiol. [2-3H]Estradiol and [16-3H]estradiol were prepared and the specificity of the labelatthe desired positionwasconfirmed aspreviously described (10). The substrate

radiohomogene-ity exceeded 98% in the case of the former, and >95% of the 3Hon themolecule was locatedat the designated

posi-tion. For the [16-3H]estradiol, substrate radiohomogeneity

exceeded 96%, with 81% of the 3H being locatedin the

a-and 15% inthe ,B-orientation. Oxidation of estradiol labeled

atboth positionsC-2 andC-16proceedswithout anisotope effect andaNationalInstitutesof Healthshift doesnot occur in the case ofthe C-2 reaction (10). Therefore, as recently

demonstrated (11-13), thereis astoichiometric relationship

between the amount of 3H released and the extent of the oxidativereactionoccurringatthat positionontheestradiol molecule. Becausethe stereospecificity of[16a-3H]estradiol

is 81%, release of 3H would represent acomposite of

16a-and 16,B-hydroxylatedmetabolites. Noattemptwasmadeto

separate the 16a- and 16,-epimers. Enzyme activity was

expressedaspicomolesof C-2or C-16oxidizedproduct (i.e.,

as reflected in 3H released) per milligram of protein per

minute.

Chickembryo liver cellsinculture. Chickembryoliver cell suspensions for culturewerepreparedasdescribed pre-viously (14). Theerythrocyte-free cellpelletwassuspended

in 75 volumes of modified F12 medium (14) supplemented

with 5% fetal bovine serum (FBS).' 5ml of the 75-fold di-lutedcell suspension,equivalentto 4-5X 105cells,wasthen addedto aflaskof25-cm2 surfacearea(Falcontissueculture

flask, type 3013; Falcon Labware, Div. Becton, Dickinson

& Co.,Oxnard, CA) and incubated for 3-4h. Atthis time, chemicals wereadded to the culture medium and the cul-tureswereincubated for afurther24h.

Allchemical agents weredissolvedin an appropriate sol-vent, as described below. The inducercompoundsemployed

in these studies comprised the following: phenobarbital (2 mM) and 2-propyl-2-isopropylacetamide (0.3- mM), pre-pared in sterile water; 3-methylcholanthrene (2

gM),

,B-naphthoflavone (15

MM),

benzo[a]pyrene (2

MM),

and

benz[a]anthracene(2

MM),

preparedinacetone; and Aroclor

1254 (2

Mg/ml),

prepared in ethanol. Noneof the solvents alone hadsignificanteffectsoncellularviabilityor on estra-diol C-2 and C-16 oxidations.

Assaysofestradiol C-2andC-16 oxidationsinlivercell

cultures. Afterincubationfor24 h inthe presenceor inthe absence of an inducer chemical, cells were rinsed with

1Abbreviation usedinthis paper: FBS, fetal bovineserum.

Earle'sbuffer and5ml of serum-free, modified F12 medium containing insulin (1 ug/ml) and [2-3H]- or [16-3H]estradiol (6.8 mCi/mmol) was added.[2-3H]Estradiol with a sp act of 20 Ci/mmol was used in the control set of experiments, which measured estradiol C-2 oxidation as a function of in-cubation period. After further inin-cubation of the cells for 2 h, 1-2 ml aliquots of medium were collected for determi-nationof C-2 or C-16 oxidation as reflected in the formation of3H20.The samples werequickly frozen in dry ice-acetone andthen lyophilized.The3H20 obtained by this procedure was collected, mixed with 5 ml of Hydrofluor liquid scin-tillation mixture (National Diagnostics, Inc., Advanced Ap-plications Institute Inc., Somerville, NJ) and the radioactiv-ity wasdetermined to within +5% accuracy with a Packard 2650 liquid scintillation spectrometer (Packard Instrument Co., Inc., United Technologies, Downers Grove, IL).

Other assays. Cytochrome P-450 was determined ac-cordingtothe method of Omura and Sato (15);an Aminco-Chance DW-2a spectrophotometer (American Instrument Co., Silver Spring, MD)wasusedfor all assays. Supernatants from a 9,000-g centrifugation of cell homogenates were pre-pared according to Sinclair et al. (16) and used for mea-surementof cytochrome P-450. Protein determinations were carried out by the method of Lowry et al. (17), with bovine

serum albuminas a standard.

RESULTS

Time course

of the oxidation of estradiol

in

liver

cell

cultures.

The formation of the 3H20 product

from both the C-2 and

C-16

oxidative

reactions

in-creased

in

the culture medium as a function of the

incubation

period

of the substrate. The time

required

for 50%

maximal

3H20

formation was -2 h for C-2

oxidation (Fig.

1

A) and

4

h

for C-16 oxidation (Fig.

1

B).

A

2-h

incubation

period

was

selected

as a rea-sonable time

point

for measurements of both

oxida-tions

in

subsequent experiments, unless otherwise

stated. The interassay results from these experiments

were

highly reproducible, the standard error of

prod-uct

formation after

incubation for 2 h

being

4.2%

for

C-2

oxidation and

4.9% for C-16

oxidation. The

stan-dard

error

of 3H20

formation obtained

upon

incuba-tion

of cultures for

24

h

was 8.5%

(six experiments)

and

7.9%

(four experiments) for the

C-2

and

C-16

ox-idations,

respectively. The standard

error

of the

mean

for product formation

among various

assays

was 1.0%

for the C-2 oxidation (six experiments) and 7.1% for

the

C-16

oxidation

(four

experiments) after incubation

for

2

h.

Data are

expressed

as

picomoles of

3H

released

per

milligram of protein per

minute.

Both

estradiol

oxidations

were

critically dependent

on

the presence

of intact liver cells and there was no

measurable

ac-tivity either

in

the

absence

of cells

or

when cells

were

denatured by

10%

trichloroacetic acid or 0.5

N

per-chloric

acid/50% methanol.

Effect of cell density

on

estradiol

oxidative

metab-olism at C-2

and

C-16. The extent

of both

the C-2

5~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IA

)

0 /~~~~~~~~~~~~~

A ₆₀

C

E 50

- 40

c3030

-20

0

6lo

0 5 10 15 20 25

Incubation Period (h)

0 04 08 12 1.6

Cell Content(mg protein /flask)

B -.,

8

E ₆₁

c U

0

(0

0D

r

4

0 5 10 15

Incubation Period (h)

0

E

x

0

20 25

FIGURE1 Estradiol oxidation as a function of incubation

periodincultured chick embryo liver cells. Cellswere

pre-pared and incubated inamodifiedF12/5%FBSmediumas

described in Methods. The medium was discarded after a

24-h incubation, and serum-free modified F12/insulin (1

,ug/ml) medium was added containing 0.5 MtCi of

[2-3H]estradiol (A)or[16-3H]estradiol (B).Eachdata point rep-resents the mean value of 2-4 determinations. The sp act

usedwas20Ci/mmol (A) and6.8mCi/mmol (B) for

[2-3H]-and [16-3H]estradiol, respectively. The final concentrations

of estradiol were 5 nM for [2-3H]- and 15 uM for [16-3H]estradiol.

sity in culture.

Although

an increase in

product

for-mation

by

these reactions was demonstrable with

in-creasing cell

density,

the amount of

product (3H20)

formed per

milligram

of protein tended to decrease

at

higher

cellconcentrations. A

plateau

ofactivity was

reached at a cell

density

of 1.5 mg

protein/25-cm2

flask for both the C-2

and

C-16reactions. These data

suggestthattheextentofmetabolismatboth positions

on the steroid molecule is influenced

by

cell

density

in culture. To avoid variation in the oxidative bio-transformations duetodifferences in cell density,

ex-perimentswere

performed

inflasks that

originally

con-tained 5 ml of a 1:75-fold suspension. This

generally

yielded

2 mg of protein per

flask

(Fig. 2, A and B).

Effects of

inducers of

cytochrome

P-450 on

estra-diol metabolism. The effects of known inducers of

cytochrome

P-450 on the oxidative metabolism of

es-tradiol at the C-2 and C-16 positions were examined

04 08 12 16

Cell Content(rmg protein/f lask)

FIGURE 2 Effect of cell density on estradiol oxidation in

cultured liver cells. Cell suspensions of 1:75, 1:100, 1:150, 1:200,and1:400 (i.e., 1volof packed cells suspendedin400

vol ofmedium)wereprepared and incubated under the

stan-dard assay conditions outlined in Methods. The extent of

estradiol oxidation was determinedas 3H20 released from

[2-3H]-(A)and

[16-3H]estradiol

(B) (15 MM, 6.8mCi/mmol).

(Table

I).

Phenobarbital and

2-propyl-2-isopropyla-cetamide have been shown to induce

cytochrome

P-450in thecultured liver cellsystem usedin

this

study

(16, 18). These

compounds significantly stimulated

C-16

oxidation,

producing

a 1.9- and

2.6-fold

increase over control

values, respectively.

C-2

oxidation

was

stimulated toa lesserextent (1.7- and 1.2-foldgreater

than control

values,

respectively). Polycyclic

aromatic

hydrocarbons

and

other compounds,

i.e.,

,-naphtho-flavone, 3-methylcholanthrene, benz[a]anthracene,

and

benzo[a]pyrene, which induce

a

specific

groupof

cy-tochrome P-450s

collectively

called P-448, did not

cause

appreciable changes

in the extent of

oxidation

at C-16. In contrast to their lack of effect on C-16

oxidation,

a

significant

enhancement ofC-2 oxidation wasobserved with

fB-naphthoflavone,

3-methylcholan-threne, benz[a]anthracene,

and

benzo[a]pyrene,

this resultedin 2.2-, 1.5-, 1.5-, and 2.2-fold increases over

In

4

7-E

C 3

c

x

0

,_,1

101

20

51

TABLE I

Effects of Inducers of MixedFunction OxidasesontheOxidative Metabolism of Estradiolat C-2andC-16

Estradiolmetabolism

C-2oxidation C-16oxidation

Ratio Ratio

Dose relative relative

Compound (concentration) Mean±SEM P tocontrol Mean±SEM Pvalue tocontrol

pmol/mg pmol/mg

protein/min protein/min

Control 47.5±1.3 4.3±0.7

Phenobarbital 2 mM 81.7±1.0 <0.001 1.7 8.0±0.1 <0.001 1.9

2-Propyl-2-isopropylacetamide 0.3MM 56.7±0.33 <0.001 1.2 10.8±0.3 <0.001 2.6

3-Methylcholanthrene 2MM 70.8±1.3 <0.001 1.5 4.9±0.2 NS 1.2

,-Naphthoflavone 15_AM 104.2±7.3 <0.001 2.2 5.8±0.3 <0.05 1.4

Benzo[a]pyrene 2 MM 104.2±2.3 <0.001 2.2 4.9±0.3 NS 1.2

Benz[a]anthracene 2 MM 72.5±0.3 <0.001 1.5 4.3±0.1 NS 1.0

Aroclor 1254 2Mg/ml 68.3±1.7 <0.001 1.4 5.7±0.1 NS 1.4

Cellswereprepared and incubatedinmodified F12/5%FBS mediuminthe presence ofaspecificchemical inducers of mixed function

oxidases. After incubation for 24 h, the medium was replaced with serum-free modified F12/insulin (1 Mg/ml) medium containing

[3H]estradiol (6.81 mCi/mmol). The cells were then incubated for another 2 h,at which timealiquotsof medium wereobtained for

evaluation of the amount of 3H20 released by the site-specific oxidation of estradiol as described in Methods. Data represent the

mean±SEMvalues offour to sixdeterminations.

The values listed for controlsamples correspond to0.070±0.002and 0.0037±0.0008 of the added substrateper milligram proteinper minute for the formationof3H20 viathe oxidation of estradiolat C-2 andC-16, respectively. Pvalues< 0.05 wereconsidered to be

statistically significant.

control

values,

respectively. The postmitochondrial

fraction (9,000 g) from

avian

hepatocytes

incubated

with

phenobarbital

or

2-propyl-2-isopropylacetamide

displayed

an

increased CO-reduced difference

spec-trum

with

a

peak

at 453.5

nm, the

cytochrome subtype

in

the

avian

liver (16, 18) that

corresponds

to P-450 in rat

liver.

Cells

treated with

3-methylcholanthrene,

,B-naphthoflavone,

or

Aroclor

1254

showed

an

in-creased

peak

at 451

nm, the

cytochrome subtype

in

the

avian

liver

that

corresponds

to P-448 in rat

liver

(Fig. 3).

Effects of

inducers

of

cytochrome

P-450 on

estra-diol

metabolism

in

chick

embryo

liver

in ovo.

Ex-periments

corresponding

to

those

in

liver

cell cultures

were

also conducted with liver

microsomes

prepared

from chick embryos

in ovo

treated

with the

same

chemicals

in

order

to

compare the metabolic

activities

of whole liver with those

of cultured liver cells from

the

same

species.

In

this experimental system, natural

steroids as well as

drugs

and other

foreign

chemicals are known to induce

cytochrome

P-450 in liver

(19,

20).

The

doses of

drugs

used

per

kilogram of body

weight

were as

follows: phenobarbital (100 mg),

benzo[a]pyrene (40 mg),

or

3-methylcholanthrene (40

mg).

The individual chemicals were

injected

through

the airsac of

17-d-old

chick

embryos

24 h

before they

were

killed. Measurements

of the extent of the

estra-diol C-2 and C-16

oxidations

were

carried out

in

vitro

on

hepatic microsomes

prepared

according to the

method described by Numazawa et al. (12). Analysis

of

3H20

was

performed

as

described for the liver cell

cultures.

Treatment

of

chick embryos with

3-meth-ylcholanthrene and benzo[a]pyrene

preferentially

in-duced

C-2

oxidation, whereas the enhancement

of

C-16

oxidation by these

two

chemicals

was

minimal.

Phe-nobarbital,

on

the other

hand,

preferentially induced

C-16

oxidation and

did not

increase C-2 oxidation

sig-nificantly (data

not

shown). These

findings

indicate

that cultured hepatocytes and whole avian liver

re-spond similarly by differential induction of C-2 and

C-16

estradiol oxidative

reactions in

response

to

chem-icals

known

to

induce distinctive species of cytochrome

P-450.

The apparent Michaelis

constant

(Km)

and

maxi-mum

velocity (Vmax) of estradiol oxidations

atC-2

and

C-16 in cultures of chick

embryo

liver cells were

de-termined. For

these

studies, the specific activity of the

substrate

estradiol

was

held

constant

(17,000

dpm/

nmol) per

assay.

A Lineweaver-Burk

plot

of

C-16

ox-idation

yielded

an

apparent Km of

30.3

,M

and a

Vmax

of 11.7

pmol/mg protein per

minute

(Fig.

4

A).

Es-tradiol oxidation

at C-2

showed

an

apparent Km

23.5

410

4535

PPIA PB -Control

:

I

490nm

10

3MC

---- _nNF

Aroclor1254

1...

w..

...;,

490 nm

450

FIGURE3 Effects of special chemical inducers of mixed

function oxidases on the induction of cytochrome P-450in

cultured liver cells. Cells were prepared and incubated as

describedinMethods. After incubation for24hwith chem-icals, culture disheswererinsed andcellswerescraped and

homogenizedin 1 ml of0.1 Mphosphate buffercontaining

20% glycerol/0.2% Emulgen 913/1 mM EDTA and 1 mM

dithiothreitol. Homogenates were centrifuged at 10,000 g

for30 min and supernatants were used for cytochrome

P-450determination by the method ofOmura and Sato (15).

3MC, 3-methylcholanthrene;

#NF,

0-naphthoflavone;

PB,

phenobarbital; PIA, 2-propyl-2-isopropylacetamide.

,iM and

a

Vmax of

119

pmol/mg

protein per minute

(Fig. 4 _B).

DISCUSSION

The

results of

the

present study

demonstrate

that cultured avian

embryo liver cells

actively oxidize

es-tradiol at the C-2 and C-16 positions and that these

reactions in avianliver

cells

appear tobe cytochrome

P-450-dependent. Furthermore,

oxidation of

this

es-trogen at either position C-2 or C-16 can be

differ-entially

stimulated

by chemicals known

to induce

dis-tinctive speciesof

cytochrome

P-450in the liver. The differential induction

of

steroidoxidative metabolism

in the cultured

hepatocytes

was also observed with

liver microsomes from

chick

embryos treated in ovo

with various

inducers of

this

hemeprotein.

Our

find--5 0 5 10 15

1 _(M)-1X104

-5 0 5 10 15

1 _(-

_il

x 0-4

FIGURE4 Lineweaver-Burk plot of estradiol oxidation. (A) C-16oxidation; (B) C-2 oxidation. Cellswereprepared and

incubated as described in Methods. The medium was

dis-carded after a 24-h incubation and serum-free modified

F12/insulin (1

jAg/ml)

medium containing [3H]estradiol

(17,000dpm/nmol) wasadded. The extentofestradiol

ox-idation wasexaminedatvaryingconcentrationsof estradiol

(3.7to -117 uM). Each point representsthe mean of four

determinations.

ings

therefore indicate

that exposure to

drugs and

other chemical inducers of distinctive forms of

cyto-chromeP-450 in livercan, tosomeextent,

selectively

alter estradiol metabolism towards the differential

for-mation of C-2 or C-16 oxidized metabolites.

Differ-ential enhancement of

androgen hydroxylases by

chemical inducers of

hepatic

cytochrome P-450 has also been demonstrated

by

Conney (1) and Conney and Klutch (21).

I

0.01 / I ::. ,

,,

_~~~~~~~~~~i,

..,~~~~~~~~~~~:

.1

410

*,

U%.jI

I

I

,/

500 T

0~~~~~~~~~~~~~~~~~

_ 400

-E

- 300

-g 200

The radiometric procedure used

in

these

studies

permits

a

ready

assessment of

the total

in vitro

me-tabolism of estradiol

in

cultured

liver cells via its two

principal oxidation pathways.

In

this

method, the

re-lease

of

3H

from radiolabeled

estradiol (10)

was

used

as a

determinant of a

site-specific

oxidation on the

estradiol

molecule. The data obtained

by

the

radio-metric technique, including varying incubation time,

protein content,

or

substrate concentration,

were

all

in

conformity with

an

enzymatic basis

forthe reactions

measured by the release of

3H

from the steroid

sub-strate.

Recently Ryan

et

al. (22), using

an in vitro

recon-stituted mixed

function

oxidase system

composed of

purified cytochrome P-450, NADPH-cytochrome

c

re-ductase,

and

lipid,

demonstrated that

cytochrome

P-450e,

which is inducible

in rat

liver

by Aroclor

1254

and phenobarbital, catalyzes

the 2-hydroxylation of

estradiol.

Our

data, utilizing

a radiometric method to

measure

the

site-specific

oxidation at

C-2,

are consis-tent

with this

finding, although

we were not able to

quantitate the

2-hydroxylated product

of estradiol be-cause

of the small

amounts

of

tissue available for

study.

However,

as

recently

shown

by

others using other

ex-perimental models (11-13),

it is a reasonable

assump-tion

that the formation of 3H2O from both

[2-3H]estradiol and

[16-3H]estradiol

in

these liver cells

reflects the formation of the expected

C-2

and

C-16

hydroxylated products.

The steroid oxidations demonstrated

in avian

liver

also represent the

principal

metabolic

biotransforma-tions

of estradiol

in

humans (23). The fact that

they

can

be induced

by

drugs

and other

foreign chemicals,

and that such substances may also

preferentially

en-hance the

C-2or

the

C-16

reaction, suggests

a

possible

mechanism

whereby

exogenous chemicals could

sig-nificantly

alter

the

profiles

of

endogenous

hormone

metabolism

in

humans. Alterations

in

these

metabolic

patterns

may

have

potential biological significance

since

many of the metabolites of such steroid hormones

as

testosterone, progesterone, and estradiol (24-29)

can

exert

potent

biological

actions

of their

own. C-2

and

C-16

oxidations

of estradiol have been

presumed

to

be

mediated

by cytochrome

P-450; the results of the

pres-ent

study

support this

view

and indicate that chemicals

known

to

induce selective species of

cytochrome

P-450,

can

preferentially

enhance either

C-2 or C-16

oxidation of the hormone.

Thus,

exposure

to

other

types of environmental chemicals, including

those

de-rived

via

the

diet, which

are

known

to

enhance

cy-tochrome

P-450-dependent

drug oxidations (30, 31),

may also result

in

alterations

in

the oxidative

metab-olism of estradiol.

Recent

studies

in

humans have

in

fact shown that alterations

in

the

protein/carbohy-drate ratio in the diet result in selective changes in the

rate

of C-2 oxidation of this hormone (32).

The

avian

embryo

liver cell culture system used in

this study has proved valuable for studying a wide

variety of metabolic processes in isolated hepatocytes,

including aspects of heme and cytochrome P-450

me-tabolism (14, 16, 18, 33-35), malic enzyme and fatty

acid

synthesis

(36, 37),

plasma protein production (38),

and

the interactions of environmental chemicals with

heme

pathway enzymes (39). The data in this report

indicate that this experimental system, when coupled

with

radiometric analysis (10), is also useful for

defin-ing

site-specific steroid hormone oxidations in liver

cells and the

effects

on

such

biotransformations

of

drugs and other chemicals

to

which humans may be

exposed.

ACKNOWLEDGMENTS

We thank Dr. Jack Fishman, The Rockefeller University, for the gift ofradiolabeled estradioltracers used in this study.

2-Propyl-2-isopropylacetamidewas providedby

Hoffmann-La Roche, Inc. The secretarial assistance of Ms. Karen Aguirre is gratefully acknowledged.

This researchwassupportedinpartbyU. S.Public Health

Servicegrant ES-01055.

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